PMC

Effect of age and gender on mRNA levels for 1,25D₃-MARRS. Duodenal mucosae were collected from each of the indicated age groups and frozen in liquid nitrogen. Total RNA was prepared with the TRIzol reagent, and 1 μg was used for RT-PCR with primers specific for 1,25D₃-MARRS protein or GAPDH. (Upper) Males. (Lower) Females. The ratios for MARRS/GAPDH are provide underneath each panel.

(A) Sequence of the full-length cDNA and translated polypeptide. Amino acids highlighted in blue correspond to the predicted signal peptide, and amino acids highlighted in yellow correspond to the N terminus of the purified protein recognized by antibody 099 and used for back translation and database searching. Consensus thioredoxin motifs are highlighted in purple. The transcript contains a long 3′ UTR after the KEDL termination sequence. (B) Schematic of targeted portion of MARRS transcript and site of ribozyme cleavage by MARRS 6 ribozyme.

Effect of age and gender on the rapid, 1,25(OH)₂D₃-mediated stimulation of phosphate transport in perfused duodenal loops. Chickens were raised on a normal, vitamin D-replete diet for 7, 14, or 28 weeks before experimentation. Arterial perfusion with GBSS containing 0.125% (wt/vol) BSA and the vehicle ethanol [0.005% (vol/vol), final concentration] proceeded for a 20-min basal period, and samples were collected during the latter 10 min. At T = 0, arterial perfusion either continued with control media (open circles) or 130 pM 1,25(OH)₂D₃ (filled circles). The lumen was perfused with GBSS lacking bicarbonate and containing 2 μCi/ml of H₃³²PO₄. Transport was measured by the amount of radioactivity appearing in the venous effluent. Results are presented as mean ± SEM (or range) for 7-week-old males (A) (n = 4 controls and 4 treated), 14-week-old males (B) (n = 5 controls and 3 treated), 28-week-old males (C) (n = 2 controls and 4 treated), 7-week-old females (D)(n = 2 controls and 5 treated), 14-week-old females (E)(n = 3 controls and 4 treated), and 28-week-old females (F) (n = 2 controls and 5 treated). ^*, P < 0.05, relative to corresponding controls for times indicated by arrows.

The 1,25D₃-MARRS protein mediates phosphate uptake and PKC activation. Isolated intestinal epithelial cells were cultured overnight without serum. The following day (A), media were replaced with GBSS/0.1% BSA without or with Ab 099 (1:500 dilution) and incubated for 5 min (23°C). Each dish then received 1 ml of GBSS containing 4 μCi of H₃³²PO₄ and vehicle (Con and cells pretreated with antibody, AbCon) or 130 pM 1,25(OH)₂D₃ (final concentration; 1,25D₃ and cells pretreated with antibody, Ab1,25D₃) and incubated for an additional 7 min. (B-E) Cells were either not transfected (No Rx) or transfected with MARRS 6 or MARRS 8 ribozymes for 5 h before addition of serum and were assayed 22 h later. (B) Cells were incubated for 7 min for ³²P uptake as in A but without the antibody preincubation. (C) Cells were incubated for 1 min with or without hormone in GBSS and then collected for PKC analyses. (D) Cell lysates were incubated with [³H]1,25(OH)₂D₃ in the absence or presence of excess unlabeled hormone. Bound and free steroid were separated by perchloric acid precipitation to determine 1,25D₃-MARRS-protein-specific binding (D) or by hydroxylapatite to determine specific VDR binding (Inset). (E) Cell lysates (30 μg per lane) were separated on SDS/PAGE and blotted onto poly(vinylidene difluoride) membranes, and Western blot analyses were performed with Ab 099 (against the N terminus of the 1,25D₃-MARRS protein) as primary antibody. Values are presented as mean ± SEM for 6-11 independent samples. ^*, P < 0.02, relative to corresponding controls.